Clostridium difficile, a gram positive, strict anaerobic bacterium, produces two toxins, toxin A (enterotoxin) and toxin B (cytotoxin) which have been implicated in the etiology of antibiotic-associated diarrhea and pseudomembranous colitis (PMC) It has been suggested that the two toxins work synergistically to induce PMC. Toxin A elicits hemorrhagic response in rabbit intestinal loop and toxin B causes characteristic rounding of cells by modifying cellular proteins that regulate the structure and function of actin cytoskeleton. The molecular and biochemical processes leading to these cytopathic effects have not been fully characterized. The major goals of this proposal are to identify the amino acids which are essential for biological activity of toxin B and to characterize its active site. Specifically, it is proposed to clone the 7.1 kb toxin B gene which was previously PCR amplified in an expression vector and express the protein in E. coli. The expressed toxin B protein will be purified and chemically modified with arginine (and other amino acids) specific reagents to identify the arginine and other amino acids residues which are essential for cytotoxicity. PCR site-directed mutagenesis technique will be used to replace the essential arginine and other amino acids. The effects of these substitutions on biological activities will be assessed. Furthermore, toxin B expressed in E. coli should be free from copurifying contaminating proteins found in clostridial cells and would be suitable for studying the quaternary structure of toxin B and the mechanisms of its cytotoxicity, such as phosphorylation of actin proteins. The investigations presented in this proposal would provide information not only on mechanisms of clostridial toxins which do not have ADP-ribosyltransferase activity but also on the dynamics of actin organization and regulation.